Stent-based delivery of AAV2 vectors encoding oxidation-resistant apoA1

In-stent restenosis (ISR) complicates revascularization in the coronary and peripheral arteries. Apolipoprotein A1 (apoA1), the principal protein component of HDL possesses inherent anti-atherosclerotic and anti-restenotic properties. These beneficial traits are lost when wild type apoA1(WT) is subjected to oxidative modifications. We investigated whether local delivery of adeno-associated viral (AAV) vectors expressing oxidation-resistant apoA1(4WF) preserves apoA1 functionality. The efflux of 3H-cholesterol from macrophages to the media conditioned by endogenously produced apoA1(4WF) was 2.1-fold higher than for apoA1(WT) conditioned media in the presence of hypochlorous acid emulating conditions of oxidative stress. The proliferation of apoA1(WT)- and apoA1(4FW)-transduced rat aortic smooth muscle cells (SMC) was inhibited by 66% ± 10% and 65% ± 11%, respectively, in comparison with non-transduced SMC (p < 0.001). Conversely, the proliferation of apoA1(4FW)-transduced, but not apoA1(WT)-transduced rat blood outgrowth endothelial cells (BOEC) was increased 41% ± 5% (p < 0.001). Both apoA1 transduction conditions similarly inhibited basal and TNFα-induced reactive oxygen species in rat aortic endothelial cells (RAEC) and resulted in the reduced rat monocyte attachment to the TNFα-activated endothelium. AAV2-eGFP vectors immobilized reversibly on stainless steel mesh surfaces through the protein G/anti-AAV2 antibody coupling, efficiently transduced cells in culture modeling stent-based delivery. In vivo studies in normal pigs, deploying AAV2 gene delivery stents (GDS) preloaded with AAV2-eGFP in the coronary arteries demonstrated transduction of the stented arteries. However, implantation of GDS formulated with AAV2-apoA1(4WF) failed to prevent in-stent restenosis in the atherosclerotic vasculature of hypercholesterolemic diabetic pigs. It is concluded that stent delivery of AAV2-4WF while feasible, is not effective for mitigation of restenosis in the presence of severe atherosclerotic disease.


Expression of apoA1 (WT and 4WF) following AAV2-mediated transduction in various cell types.
To analyze the transduction capacity of AAV2 vectors containing apoA1(WT) and apoA1(4WF) cassettes under control of CMV promoter, Raw 264.7 murine macrophages, rat primary aortic smooth muscle cells (RASMC), human embryonic kidney cells (HEK-293) and rat blood outgrowth endothelial cells (BOEC) were transduced with AAV2-apoA1(WT and 4WF) vectors at multiplicity of infection (MOI) of 10 5 -5 × 10 5 (10 10 -3.5 × 10 10 VG/ml). The conditioned media containing lipoprotein free FBS were collected 3 days posttransduction, and the secreted apoA1 variants in the media were determined by ELISA. HEK-293 and BOEC exhibited higher transducibility than SMC, while apoA1 concentrations in the media conditioned by Raw 264.7 macrophages were very low (Fig. 1A). The abundance of WT and 4WF variants in the conditioned media of HEK-293 cells was similar, while in all other cell types, it differed 2-4 fold (WT > 4WF in BOEC, and WT < 4WF in SMC and Raw 264.7). Collectively these results demonstrate that BOEC, and SMC have capacity for AAV2mediated transduction and apoA1 production. To further confirm the expression of apoA1 variants in the transduced cell lines, rat aortic endothelial cells (RAEC) and RASMC were transduced with AAV2-apoA1 (WT and 4WF) as above and apoA1 (WT and 4WF) expression was confirmed by immunofluorescence. As negative controls, non-transduced RAEC, and RASMC omitted anti-human apoA1 antibody exposure were used. While only light staining was detected in the control groups, AAV2-apoA1(WT)-and AAV2-apoA1(4WF)transduced cells exhibited distinct cellular staining, confirming apoA1 expression in these cells (Fig. 1B). The staining intensity was higher in RAEC than in RASMC and was comparable between the cells transduced with AAV2-apoA1(WT)-and AAV2-apoA1(4WF) collaborating ELISA results.

Cholesterol efflux properties of WT and 4WF apoA1 at baseline conditions and in a pro-oxidative environment.
To study cholesterol efflux to the medium conditioned by AAV2-apoA1(WT)-and AAV2-apoA1(4WF)-transduced HEK-293 cells, the collected media were concentrated to 10 µg/ml to match the concentration of recombinant human ApoA1 preparation (rh-apoA1). The media were either used directly in the cholesterol efflux assay or were exposed to escalating concentrations of hypochlorous acid (HOCl) to emulate oxidative stress. The conditioned media from AAV2-apoA1(WT)-transduced cells showed a 27% higher efflux capacity than media from AAV2-apoA1(4WF)-transduced counterparts under basic non-oxidative conditions (HOCl/apoA1 = 0) (Fig. 2). However, rhapoA1 and apoA1(WT) lost 22%, 33% and 59%, and 51%, 64% and 72% of their efflux capacity, respectively at 2, 8 and 16 HOCl/apoA1 molar ratios (Fig. 2). In contrast, the 4WF variant of apoA1 displayed only a 11%, 17% and 26% decrease of its efflux capacity at the same conditions (p < 0.05 for all comparisons between 4WF and both rhapoA1 and WT), therefore indicating oxidative stress resistance of this construct (Fig. 2).
To evaluate the effects of apoA1 transduction on inflammatory activation of endothelium, rat monocytes isolated from peripheral blood were fluorescently tagged with PKH-26 dye. 5 × 10 4 monocytes were then added to the wells with TNFα-stimulated non-transduced RAEC (NT) or RAEC transduced with AAV2-apoA1(WT) and AAV2-apoA1(4WF). Monocytes incubated with NT BOEC exhibited significantly higher attachment when compared to BOEC transduced with WT and 4WF (22% and 33%, respectively; p < 0.001 for both comparisons) (Fig. 3F). Inhibition of monocyte attachment to AAV2-apoA1(WT)-and AAV2-apoA1(4WF)-transduced RAEC monolayers was not caused by AAV2 transduction-related cell signaling since eGFP-transduced RAEC demonstrated a trend to higher monocyte binding (p = 0.067) than NT RAEC (Supplemental Fig. 4).   Acute in vivo studies-AAV2 stent loading. RT-PCR with AAV2-specific primers was used to determine the number of viral genomes associated with the surface of undeployed stents, stents retrieved from the coronary arteries of the experimental animal 1 h after implantation, and the arterial tissue directly contacting the explanted stents. 1.38 × 10 8 ± 3.71 × 10 7 of AAV2 genomes were found in association with the control stents that were not deployed in the animals. The stents explanted at 1 h post-deployment displayed 1.59 × 10 7 ± 3.1 × 10 6 viral genomes (i.e., approximately 12% of the initial loading). Finally, 3.65 × 10 5 ± 1.97 × 10 4 AAV2 genomes (0.26% of the total vector dose) were transferred to the arterial wall (Fig. 4C).
In vivo reporter studies. Arterial segments opposing the implanted AAV2-eGFP stents were harvested 7 days after stent deployment in the coronary arteries of healthy pigs and immediately processed for Western blot studies. Western blot analysis of AAV2-eGFP GDS-treated arteries ( Hypercholesterolemic diabetic swine studies. The prevalence of neutralizing anti-AAV2 antibodies in the plasma of study animals. Blood was collected for measuring titers of AAV2-neutralizing antibodies at the inception of the study, 1 week prior to surgical intervention and at euthanasia. Four out of 13 animals tested positive for the preformed anti-AAV2 antibodies at the beginning of the study. These animals were, therefore, either removed from the study or assigned to the BMS group (Supplemental Fig. 6). Antibody titers either remained unchanged or increased insignificantly after the 24-week period of model development (Fig. 6A). In contrast, plasma of all 9 assayed animals harvested when sacrificed exhibited high levels of neutralizing antibodies exceeding the cut-off values for the assay positivity (more than 50% decrease of transduction at 1:20 plasma dilution compared to the reference control of no plasma supplementation) (Fig. 6A). (C) Immobilized AAV2 particles were disengaged from the not deployed stents, from the stents deployed in the pig coronary arteries and extracted from the arterial tissue underlying deployed stents. The viral DNA was isolated using a QIAamp DNA minikit and amplified in RT-PCR reaction with AAV2-specific primers. The number of AAV2 genomic copies was quantified using a calibration curve constructed with a known amount of AAV2 genomes. Human apo1(4WF) expression after implantation of AAV2-apoA1(4FW)-eluting GDS. Expression of human apoA1(4WF) isoform in the porcine arterial tissue located within 2-3 mm of the implanted stents was found in 5 out of 12 analyzed AAV2-apoA1(4FW)-eluting GDS. Low expression levels were exceeded baseline 2.6-8-fold (Supplemental Fig. 7).

Discussion
The studies reported herein established that AAV-mediated gene therapy with human apoA1(4WF) transgene is capable of rescuing the attenuated efflux of cholesterol from macrophages under physiologically-relevant conditions of oxidative stress. Furthermore, site-directed mutagenesis resulting in four Trp-Phe substitutions in the apoA1(4WF) isoform does not compromise the inherent apoA1 ability to inhibit proliferation of SMC, promote proliferation of endothelial cell lineage, and reduce inflammatory activation of the endothelium. Vascular tissue transduction by AAV2 vectors immobilized on the stent struts was shown to result in short-term expression of the delivered transgene. However, prolonged transduction is compromised by low initial transduction of vascular tissue, pre-existing and, conceivably, by newly induced anti-AAV2 neutralizing antibodies, undermining ultimate therapeutic effects. Further refinements of apoA1(4WF) GDS are needed for potential clinical use.
The potential role of augmenting apoA1/HDL quantity and functionality in the prevention of in-stent restenosis, late stent thrombosis, and neoatherosclerosis. Re-occlusion of stented arteries with neointimal tissue (ISR), a thrombus (LST), or unremitted expansion of atherosclerotic plaque in the culprit stented lesions (NA) remains the central unresolved problem of interventional cardiology. One of the main limitations of the DES devices is their indiscriminate growth-inhibiting effect towards both SMC and endothelium, resulting in the escalation of thrombotic complications. ApoA1 and HDL (as the physiological carrier of apoA1) are unique in their opposing actions on SMC and endothelium. Previous studies demonstrated that both apoA1 and reconstituted HDL, in physiological concentrations, decrease proliferation of TNFα-stimulated human umbilical SMC 33 while increasing survival and proliferation of endothelial cells 34 and endothelial precursors 35 . Likewise, apoA1 and HDL were shown to promote endothelial cell migration in vitro 36 , support the restoration of the integral endothelial monolayer in the mouse model 36 , and inhibit SMC migration 37 , thus limiting the number of media-originated SMC in the intimal space.
In addition to the modulation of proliferative and migratory characteristics of SMC and endothelial cells, the beneficial, pro-healing effects of apoA1/HDL in the stented arteries include: increased efflux of cholesterol from foam cells 9 , enhancement of nitric oxide production and bioavailability 38 , mitigation of reactive oxygen species damage 39 , anti-thrombotic activity 40 and anti-inflammatory effects 41 . Collectively, these traits make apoA1 an appealing choice for the prevention and treatment of vascular pathology precipitated by stenting 10,42 . Gene delivery stents. As autonomous implantable devices, DES possess a limited loading capacity that is often inadequate to provide therapeutic concentrations of drug for the entire period of post-angioplasty vessel remodeling. Conversely, stents furnished with gene delivery vectors immobilized on the surface of the struts, i.e., gene delivery stents (GDS), may overcome this intrinsic DES deficiency by delivering the gene vector into the stented vessel wall and making the vascular tissue a permanent production site of the encoded therapeutic protein 43 . We 44 and others 45 showed sustained transgene expression in mammalian vasculature upon transduc- Advantages of apoA1(4WF) over apoA1(WT) in cholesterol efflux under conditions of oxidative stress. Efficient cholesterol efflux in stented patients is associated with a reduced need for reintervention related to ISR 19 and NA 20 . Platelet aggregation, which underlies LST on the incompletely endothelialized stent surface, is also reversely correlated with cholesterol efflux values 46 . Cholesterol-mobilizing properties of apoA1 are severely impaired by oxidative modification of several key Trp residues 23 . To this end, the oxidation-resistant 4WF apoA1 mutant, in which vulnerable Trp were substituted for Phe, exhibited greater cholesterol efflux than a WT protein in a pro-oxidative environment 21,24 . In our studies, apoA1(4WF) endogenously produced in AAV2-apoA1(4WF) transduced cells was capable of maintaining cholesterol efflux at the most severe conditions of oxidative stress, while the endogenously produced, or exogenously supplemented, WT apoA1 demonstrated a significant reduction of the efflux activity compared to the non-oxidative environment (Fig. 2). It is noteworthy that previously demonstrated oxidation resistance of apoA1(4WF) produced in the prokaryotic expression system 24 , would not necessarily warrant the effectiveness of the apoA1(4WF) produced in the eukaryotic cells, since post-translational modifications affect apoA1 functionality 47 . cholesterol efflux assay are mechanistically related to the apoA1 effects on the proliferation and migration of SMC and BOEC and the anti-inflammatory properties of apoA1. We therefore investigated the competence of apoA1(4WF) to exhibit beneficial physiological effects on the primary cell types involved in the pathogenesis of stenting-related complications. We showed a profoundly decreased growth rate of the primary vascular SMC transduced with both AAV2-apoA1(WT) and AAV2-apoA1(4WF) compared to the non-transduced SMC (Fig. 3A), with no significant difference between the two forms of apoA1, and an increased proliferation in AAV2-apoA1(4WF)-transduced BOEC cultures compared to their non-transduced counterparts (Fig. 3B).
Since cell quantification assays reflect the combined effects of proliferation and apoptosis on cell growth, the WST-8 results were further corroborated with Ki67 immunostaining which is exclusively associated with cell proliferation (Supplemental Fig. 2). Together, these results in AAV-transduced cells confirm the previously observed modulation of SMC 33 and EC 34 proliferative activity with exogenously supplemented apoA1(WT) and indicate a non-inferiority of apoA1(4WF) compared to apoA1(WT) as a therapeutic agent capable of simultaneous inhibition of SMC proliferation and stimulation of EC re-growth. In contrast to published data 36,37 , we were unable to find any significant impact of either apoA1(WT) or apoA1(4WF) on the migration of SMC and BOEC (Fig. 3C, D). In addition to the different contexts of apoA1 presence in the cell culture (AAV2-driven endogenous expression versus direct exogenous supplementation), this discrepancy may be related to the different methodology 37 used to measure cell migration.
We also demonstrated the attenuated intracellular ROS production, both under basic conditions and after TNFα stimulation, in AAV2-apoA1(WT)-and AAV2-apoA1(4WF)-transduced EC compared to the non-transduced control (Fig. 3E). ROS mitigation in the apoA1-expressing cells could be contributing to the decreased inflammatory activation of EC upon TNFα stimulation, as evidenced by reduced attachment of fluorescentlylabeled syngeneic monocytes to the confluent EC monolayers in vitro (Fig. 3F). These apoA1 activities were previously demonstrated only after supplementing apoA1 41 , or its peptide analog 39 , but never following endogenous production of apoA1 in transduced cells.
Reporter and therapeutic studies in porcine models. Transduction of porcine peripheral arteries following local intravascular delivery of AAV serotypes 2 and 9 was shown before 48,49 , however to the best of our knowledge, no prior study in pigs has investigated arterial gene transfer by the stent-immobilized AAV vector. To characterize gene transduction of the arterial wall with GDS in the pig model, we used the implantation of AAV2-eGFP eluting stents in the coronary arteries of healthy young animals. eGFP expression was detected in all arteries explanted 7 days after deployment of GDS (Fig. 5 A), albeit in different amounts (Supplemental Fig. 5B).
In a limited set of therapeutic studies comparing the anti-restenotic effectiveness of AAV2-apoA1(4WF) stents with AAV2-eGFP and BMS controls, we were unable to find a superior performance of AAV2-apoA1(4WF)eluting GDS (Fig. 6 B and C). Analysis of the reasons for this therapeutic failure led us to the detection of high anti-AAV2 antibody neutralizing activity in our experimental animals ( Fig. 6 A), even before the iatrogenic AAV2 exposure. At the conclusion of the experiments, at 1 month after deployment of the AAV2-eluting GDS, all animals exhibited the AAV2-neutralizing antibody titers that exceeded the widely accepted 50 cut-off of 50% reporter transgene activity inhibition in vitro. In a previous study, 30% of pigs were found seropositive for AAV2specific neutralizing antibodies 51 . This number is close to 40% observed in the current experimental series. While AAV seroprevalence in the human population 52 clearly presents a formidable hurdle for the realization of the therapeutic potential of AAV2-carrying GDS, several recent reports suggest potential approaches for mitigating the detrimental antibody responses using preemptive systemic administration of empty "decoy" AAV vectors 53 or preemptive plasmapheresis with AAV antibodies-depleting column 54 . Furthermore, the use of AAV2 vector pseudotyped with serotype 9 capsid proteins (AAV2/9) may be advantageous due to less prevalent preformed neutralizing antibodies 55 and higher transgene expression 44 compared with AAV2. In addition to the high titers of neutralizing antibodies, suboptimal transduction of the vasculature with AAV2 vector even in the absence of AAV2-neutralizing activity in the plasma is a major reason for the failure to demonstrate therapeutic effects of GDS. Additionally, attenuation of the CMV promoter activity 56 may contribute to the low levels of apoA1(4WF) detected in the vascular tissue adjacent to the deployed AAV2-eluting GDS (Supplemental Fig. 7) and the lack of the anti-restenotic effects (Fig. 6 B and C). To this end, substituting the CMV promoter with the native human apoA1 promoter reinforced with four apoE enhancers was previously shown to sustain therapeutic levels of apoA1 expression for more than 6 months in vivo 57 . Limitations. There are several significant limitations of the studies reported here. First, while the cholesterol efflux studies showed the superiority of apoA1(4WF) over apoA1(WT) under simulated conditions of oxidative stress, the experimental design chosen for the proliferation, migration, and inflammation assays used a nonoxidative environment, thus establishing non-inferiority rather than the superiority of apoA1(4WF). Second, no neutralizing antibody assays were pursued in the reporter pig studies. This reduces scientific rigor from the interpretation of the mismatch between successful reporter expression at 7 days and the lack of anti-restenotic effects at 28 days post-stenting as a consequence of escalating production of AAV2 neutralizing antibodies in the experimental animals. Third, since no therapeutic benefit of apoA1(4WF)-eluting stents was demonstrated in our studies, the anti-restenotic effectiveness of AAV2-apoA1(WT)-counterparts was not investigated in the animal model. These issues are the subject of our ongoing and planned work.

Conclusions
These studies demonstrated that AAV2-mediated apoA1(WT) and apoA1(4WF) transduction of the cell types relevant for the pathogenesis of ISR selectively modifies the physiology of SMC and EC, promoting anti-restenotic responses. Cholesterol efflux from the foam cells, hypothetically a crucial process in ISR and NA prevention, is better sustained with apoA1(4WF) than apoA1(WT) over-expression under conditions of MPO-triggered oxidative damage to apoA1 which is prevalent in CVD patients 23 . While GDS formulated with AAV2-eGFP attain transgene expression in the underlying healthy porcine arterial tissue 7 days after stent implantation, AAV2-apoA1(WT) stents implanted in the atherosclerotic arteries of the HDS model did not decrease the severity of ISR, presumably due to the intrinsically low AAV2-mediated transduction of atherosclerotic arteries and the development of dominant humoral immune response against the gene vector. Nevertheless, the prominent antiatherosclerotic and anti-restenotic properties of apoA1(4WF) warrant further investigations of its potential as a therapeutic moiety in conjunction with gene delivery stents. Cell culture. All primary cells were used in passages 3 to 7. RAEC and rat BOEC were grown in EGM-2 medium. All other cells were maintained in DMEM supplemented with 10% FBS (Gemini) and 1% antibiotic/ antimycotic mixture (Gibco). When appropriate, the medium was switched to respective basal medium supplemented with lipoprotein-free FBS (Kalen Biomedical) to avoid the effects of HDL and apoA1 derived from serum on experimental endpoints.
Cholesterol efflux experiments. HEK 293 cells were seeded in a 24-well plate. Upon reaching 75-85% confluence, the triplicate wells were transduced with AAV2-apoA1(WT), AAV2-apoA1(4WF) (both at MOI of 10 5 ) or were left untransduced. Forty-eight hours after transduction the medium was changed for unsupplemented DMEM. After 24 h, the medium was collected and concentrated 20-fold using centrifugal concentration devices with a 3 kDa cut-off membrane. The concentration of human apoA1 in each preparation was then deter-Scientific Reports | (2022) 12:5464 | https://doi.org/10.1038/s41598-022-09524-y www.nature.com/scientificreports/ mined by human apoA1 ELISA (R&D Systems) per manufacturer instructions. ApoA1 concentration in the conditioned media originated from the AAV2-apoA1(WT)-and AAV2-apoA1(4WF)-transduced cells was then adjusted with unsupplemented DMEM to 10 µg/ml. Likewise, the media from untransduced cells was spiked with the commercial recombinant human apoA1 to 10 µg/ml concentration. Murine Raw 264.7 macrophages in a 12-well plate format were treated with 0.5 µCi 3 H-cholesterol (Perkin-Elmer) formulated in sterile culture media with the addition of 0.15 µM acetylated human LDL and incubated for 48 h to allow ample cholesterol uptake by macrophages. Twenty-four hours before testing, 8Br-cAMP was added to the medium at the final 0.15 mM concentration to activate the ABCA1 production. Immediately before starting the cholesterol efflux phase of the experiment, concentrated conditioned media from HEK-293 cells containing 10 µg/mL of apoA1(WT), 10 µg/mL apoA1(4WF), or 10 µg/ml of recombinant human apoA1 spiked to the media from untransduced cells were mixed with increasing concentrations of hypochlorous acid (0, 2, 8, and 16:1 molar ratio to apoA1). ApoA1 oxidation reaction was run in triplicate aliquots at 37 °C for 1 h. Raw 264.7 cells were then carefully washed with PBS and incubated with the differently oxidized HEK 293-conditioned DMEM in the cell culture incubator for 4 h. The media were then collected and centrifuged to exclude cell debris. Cells were washed with PBS, scraped, resuspended in PBS. 5 mL of Ecolite + scintillation fluid was added to each media or cell containing scintillation vial and vortexed. 3 H count rates were then determined by using a scintillation counter (Beckman-Coulter LS6500). The 3 H-cholesterol efflux was calculated as a percentage of 3 H in the media over the total 3 H-cholesterol content in both the media and cells.
Proliferation assay. RASMC and rat BOEC grown to 70-80% confluence in T-75 flasks were transduced at MOI 10 5 (10 10 VG/ml) with AAV2-apoA1(WT), AAV2-apoA1(4WF), AAV2-eGFP or left untransduced. Four days after transduction, the cells were trypsinized, collected by centrifugation, mixed with FBS/DMSO, aliquoted and frozen. The single aliquots of non-transduced (NT), AAV2-eGFP-transduced, AAV2-apoA1(WT)transduced, and AAV2-apoA1(4WF)-transduced cells of both types were then reseeded into the 96-well plates (N = 4 wells per group; 5 × 10 3 cells/well across all experimental conditions). A group of wells in each plate was seeded at a higher density (10 5 cells/well) to achieve immediate confluence. The cells were maintained in DMEM supplemented with 10% lipoprotein-free FBS (Kalen). 20 ng/ml of rat TNFα was added to RASMC cultures to emulate the atherosclerotic milieu. The relative cell number for each transduction type was determined at days 2 and 4 with WST-8 assay by normalizing the optical density values for each well of growing cells to that of 100% confluent reference wells. Cell growth kinetics were expressed as the percent of a monolayer confluency. At the completion of the WST-8 assay at day 4 post-seeding, the cells were fixed with 10% formalin and immunostained with rabbit anti-Ki67 antibody/goat anti-rabbit AlexaFluor 555-labelled antibody and counterstained with Hoechst 33,342.
Migration assay. RASMC and rat BOEC pre-transduced en masse with AAV2 apoA1(WT), AAV2-apoA1(4WF), and frozen as detailed above for the proliferation assay, were seeded in a 48-well plate and cultured for 3-4 days in the respective media supplemented with 10% lipoprotein-depleted FBS until confluent. The cells were then starved for 36 h in media containing 0.5% lipoprotein-depleted FBS. A linear scratch injury was then inflicted to each well with a 200 µl pipette tip. The cells were washed with PBS to remove debris and imaged at 40 × magnification immediately after the scratch injury and 24 h after. The closure of the gap by inwardly migrating cells was quantitated using Image J (v1.53a).
Monocytes were isolated from 10 ml of heparinized blood harvested from naïve male Sprague-Dawley rats by Ficoll-Paque gradient centrifugation with subsequent magnetic immunoseparation using a cocktail of anti-CD8, anti-CD5, anti-CD45RA, and anti-pan T cell antibodies 61 . Isolated monocytes were then fluorescently labeled with PKH-26 dye (Millipore-Sigma, St. Louis, MO, USA) as directed by a manufacturer. 5 × 10 4 fluorescentlylabeled monocytes were then added to each well with differently transduced, TNFα-activated rat endothelial cells. Following 30 min incubation in the cell culture incubator, monocyte adhesion was examined by fluorescence microscopy. The number of monocytes attached to the activated RAEC monolayers was derived from the 100 × magnification images of the central area of each well.
To eliminate the possibility that cell signaling events triggered by RAEC transduction with AAV2 vector decrease monocyte adhesion to the endothelial monolayer, in a separate experiment PKH-26 labeled rat monocytes were added to the wells of AAV2-Egfp-transduced and non-transduced RAEC stimulated with 40 ng/ml rat TNFα (N = 4 per condition).

Quantification of AAV2 load on stents and in the arterial wall.
The extra AAV2-carrying stents not used in the animal experiments (N = 3), as well as stents (N = 3) harvested from the animal that died within 1 h of the stenting surgery, were carefully cut into 1-2 mm fragments. Arterial tissue segments (N = 3) underlying the stents harvested from the pig coronaries were separated from the metal struts and processed separately. Specifically, four fragments were excised from the central part of each arterial segment and pooled. The wet weight of the pooled specimens was ~ 30 mg. The tissue was minced using stainless steel beads and a Bullet Blender (both from Next Advance, Troy, NY). All specimens were then individually processed using QIAamp DNA minikit (Qiagen) to isolate the viral DNA. Calibration curve samples spanning 10 4 -10 10 AAV2 genomes were prepared from the stock solution of the vector. An RT-PCR reaction was then carried out with AAV2-specific primers, thus providing direct quantification of viral genomes associated with each specimen. The fractions of the AAV2 load associated with the retrieved stents and the underlying arterial tissue were then calculated, assuming the viral load of undeployed stents as 100%. To verify completeness of viral DNA extraction from the stents, a second round of DNA extraction from the already processed samples was attempted in the preliminary experiments, yielding no DNA.
AAV2-GFP stent reporter study. To study transduction of arterial tissue with AAV2-carrying stents in a pig model, 4 Yorkshire domestic pigs (22-28 kg) of both genders received AAV2-eGFP stents in the left anterior descending (LAD) and the circumflex (Cx) coronary arteries. The animals were euthanized 7 days after the surgery, and the harvested stented arteries (N = 8) were snap-frozen in liquid N 2 , pulverized under liquid N 2 , and the tissue powder was suspended in 500 µl of T-Per buffer (Thermo Scientific) supplemented with protease inhibitors (Roche), incubated on ice for 30 min, and centrifuged at 10,000 G for 10 min. Protein concentration in the supernatant of each sample was determined by the BCA assay. Fifty µg protein samples were resolved on a 4-12% NuPAGE™ Bis-Tris polyacrylamide gel, blotted to a nitrocellulose membrane, blocked with 5% dry milk/ PBS and consecutively reacted with anti-eGFP antibody (Rockland, 1:7000 dilution) and anti-β tubulin antibody (GeneTex, 1:5000 dilution), peroxidase-conjugated goat anti-rabbit antibody (Santa Cruz; 1:2000 dilution) and SuperSignal™ Pico Plus luminescent substrate (Thermo Scientific). The signal was detected using a Luminescence detection station (IVIS Spectrum) and analyzed with Image J software (v1.53a).
Hypercholesterolemic/diabetic pig model and pig stenting experiments. All animal experiments were pre-approved by the Institutional Animal Care and Use Committee of the University of Pennsylvania, carried out in accordance with federal regulations and reported in accordance with ARRIVE guidelines. To test the therapeutic effectiveness of stents formulated with AAV2-apoA1(4WF) vector immobilized on the bare metal stent struts, a hypercholesterolemic/diabetic pig model 62 was used. Before instituting model-related pharmacological and dietary interventions, all animals were screened for the presence of preformed anti-AAV2 antibodies, and the animals tested positive were either excluded from the study or were ascribed to the group treated with BMS (Supplemental Fig. 6). Totally eleven 10-12 week-old female Yorkshire domestic pigs (22-28 kg) were made diabetic by an intravenous injection of Streptozotocin (125 mg/kg). After verification of persistent hyperglycemia (> 250 mg/dl) the animals were administered a hypercholesterolemic diet (0.5% cholesterol, 5% lard, 1.5% sodium cholate) for 24 weeks. Total blood cholesterol and blood glucose levels were monitored throughout the study. If blood glucose exceeded 400 mg/dl, insulin was administered as needed.
All study animals underwent cardiac catheterization with coronary angiography. Bare metal stents, AAV2-eGFP stents, and AAV2-apoA1(4WF) stents (all 18-mm length, mounted onto 3 mm or 3.5 mm balloon catheters) were implanted in proximal and/or distal locations of each animal's left anterior descending (LAD) and the circumflex (Cx) coronary arteries (2-4 stents per animal). An inflation pressure of 10-14 atm was applied to deploy stents to achieve a 1.1-1.2 stent/artery diameter ratio. Two animals died because of ventricular fibrillation within 1 h of stenting (Supplemental Fig. 6). The harvested arteries of one of the deceased animals were used to quantify the vector load on the stents and the vector loss during deployment and the initial phase of the release. All survived animals were euthanized 4 weeks after stent deployment by IV injection of KCl (125 mg/ kg). The hearts were harvested. The stented arterial segments were excised preserving the 2-3 mm non-stented flaps of arterial tissue on both sides and flushed with heparinized saline. The stent-free overhangs were then dissected and snap-frozen in liquid nitrogen for RT-PCR studies. The stented portions of the arteries were fixed in 10% buffered formalin, methyl methacrylate-embedded, sectioned, deplastified and stained according to the Verhoeff-van Giesson method. Five sections cut 3 mm apart from each other through the entire length of the stented segment were stained and analyzed. Ends of the stented segments were excluded. Digital images of the stained arterial sections were captured at 20 × magnification, and the areas of the lumen, internal, and external elastic laminas were measured to derive the extent of restenosis, expressed as a neointima-to-media area ratio, % of luminal stenosis and neointimal thickness. These restenosis indices from 5 individual sections were averaged and the mean values were used for comparison between the different treatment groups.

AAV2-neutralizing antibodies titering.
To determine the prevalence of AAV2 neutralizing antibodies in the serum of experimental animals, blood sampled at the beginning of the study, 1 week before the intervention, and at the sacrifice was allowed to clot and centrifuged at 1500G for 15 min. Obtained sera were stored at −80 °C prior to analysis. HEK-293 cells grown in the 96-well plates to 80% confluence were transduced with AAV2-eGFP at MOI of 10 5 in the presence of 1:20 diluted serum samples or without the addition of the sera. The ensuing transgene expression was determined by fluorimetry at 485/538 nm 3 days after transduction. 50% or higher inhibition of transgene expression manifested as reduction of eGFP fluorescence intensity, denoted presence of neutralizing antibodies in the serum. The animals that exhibited the presence of neutralizing antibodies at the beginning of the study were excluded from the study or slated to the BMS treatment group.
Statistical methodology. Data are presented as means ± SD, unless specified otherwise. Differences between the groups were analyzed by ANOVA followed by a post-hoc Tukey's test, and were termed statistically significant at p < 0.05.